Figure 5

Y-box binding protein 1 (YB-1) binds to specific sites within the epidermal growth factor receptor (EGFR) promoter. (a) Sequence of the EGFR2a oligonucleotide used in the gel shift assays (-979 to -934). Highlighted sequences are the potential YB-1 binding sites. The substitutions made in the two mutants are given under the wild-type sequence. (b) Direct evidence for YB-1 binding to the EGFR promoter using gel shift assays. Nuclear extract from SUM149, MDA-MB-468 or HCC1937 cells were incubated in the presence of the EGFR oligonucleotide spanning -979 to -934. There was no binding in the absence of protein (lanes 1, 5 and 10), whereas the addition of the nuclear extract (lanes 2, 6 and 11) resulted in strong binding that could be inhibited with the unlabelled oligonucleotide (lanes 3, 7 and 12). The addition of a YB-1 antibody caused a supershift (lane 4, 8 and 13) that did not occur when the non-related CREB antibody was used (lanes 9 and 14). (c) Nuclear extracts from 6 primary BLBC samples were pooled and used in a gel shift assay for the EGFR 2a site. Lane 1 contains EGFR2a biotin-labelled oligo only. Binding to the probe is evident in lane 2, which was competed off in lane 3 and supershifted with a YB-1 antibody in lane 4. A CREB antibody was used to demonstrate specificity of the supershift (lane 5). (d) Validation of putative YB-1-responsive elements on the EGFR promoter. SUM149 nuclear extracts were incubated with either wild-type (lane 1) or mutant biotin oligo nucleotides (lanes 3, 4, and 5). A competition reaction was carried out against the wild-type (lane 2). nuclear extract bound to the wild-type sequence (lane 1), but was unable to bind the mutants (lanes 3, 4 and 5).